The long term goal of this research project is to thoroughly define the biosynthetic pathway of alginate biosynthesis, in terms of its individual steps and enzymology. These studies will lead, eventually to inhibitors for enzymes of the pathway that may be useful as drugs in the treatment of mucoid Pseudomonas infections in cystic fibrosis patients. During the period of the proposed work, we will focus our efforts on several key portions of the pathway: mannuronan C5-epimerase, GDP-manuronate polymerase, the alginate acetylation steps, and the early metabolite fluxes into the alginate pathway. These studies will serve as a foundation for future design of pathway-specific inhibitors and for sequencing and structure determination of the enzymes. Studies will in all cases by comparative between Pseudomonas aeruginosa and the prototype alginate producer, Azotobacter vinelandii. Our specific aims, listed in order of priority, during this funding period will be: (1) Isolation, purification, and characterization of mannuronan C5- epimerase from Azotobacter vinelandii and Pseudomonas aeruginosa (cystic fibrosis isolates), and conducting initial studies of its active site. (2) Studies of internal isotope return in the epimerase to determine whether a one- or two-base catalytic mechanism is operating. (3) Further development of our model for polymer-level control of epimerase action pattern, including binding studies in the presence of various metal ions. (4) NMR/molecular modeling studies of metal ion complexes with alginate components to elucidate conformational dictates of the epimerase. (5) Elucidate the functions of the O2 and O3 acetyl substituents in bacterial alginate. (6) Elucidate the location and polymerization mechanism of GDP-mannuronate polymerase, and identify the initiation carrier molecule. (7) Survey, through stable isotope/NMR methods, primary metabolic fluxes feeding into the alginate pathway.